This invention relates to a novel N-acetyl hexosamine-dehydrogenase (hereinafter referred to as N-AHDH) which acts upon N-acetylglucosamine or N-acetyl galactosamine to convert them to N-acetylglucosamino lactone or N-acetylgalactosaminolactone and, at the same time, reduces the oxidized form of nicotinamide adenine dinucleotide (NAD.sup.+), to the reduced form of nicotinamide adenine dinucleotide (NADH). This invention relates also to a process for producing said N-AHDH, an enzymatic method for quantitatively analyzing N-acetylglucosamine or N-acetylgalactosamine by the use of N-AHDH, and a kit for use in the quantiative analysis.
2. Description of the Prior Art
Since the discovery of the fact that complex sugars or glycoconjugates present in the surface layer of cells and body fluids in combination to protein or the like carries the informations for controlling living body, the studies of complex sugars or glycoconjugates has made a rapid progress. As the result, the relation between abnormal control of living body and structural abnormality of complex sugars or glycoconjugates has gradually been elucidated.
On the other hand, mucopolysaccharides are sometimes excreted into urine largely or accumulated in tissues due to the metalbolic abnormality of mucopoly-saccharides of complex sugars, so that it is important to study the structure of these sugars in order to specify its cause.
In clinical tests, enzymatic activity of N-acetylglucosamine metabolic system, i.e. the activities of .beta.-N-acetylglucosaminidase and lysozyme, are actively measured in order to specify the extent and location of disorder in kidney.
In these studies and measurements of enzyme activity, quantitative analysis of N-acetylglucosamine plays an important role. Thus, an excellent method for its measurement has been desired among specialists in the art.
Generally, quantitative analysis of N-acetylglucosamine is carried out by chemical methods such as Morgan-Elson method, etc. However, methods using an enzyme are superior in accuracy and simplicity. As one example, the method of Japanese Patent Application Kokai No. 59-156299 using N-acetylhexosamine-oxidase can be referred to. According to this method, N-acetylhexosamine is quantitatively analyzed by reacting N-acetylhexosamine with N-acetylhexosamine-oxidase and determining the resulting product such as hydrogen peroxide and the like or by determining the quantity of oxygen absorbed with progress of the reaction.
However, since N-acetylhexosamine-oxidase has a relatively broad substrate specificity, it exercises an action upon N,N'-diacetylchitobiose and the like, too. Since this sugar forms two molecules of N-acetylglucosamine upon one reaction with .beta.-N-acetylglucosaminidase, it is usable as an excellent substrate for use in high sensitivity measurement of enzyme activity. However, it has been unusable as a substrate for measurement of .beta.-N-acetylglucosaminidase in urine for the above-mentioned reason.
It is also known that the reductive substances present in urine exercise an influence on the quantitative analysis system of hydrogen peroxide, and accuracy of measurement is somewhat deteriorated by it. However, it is known that, in case of quantitative analysis system of NADH, these substances hardly exercise such an influence. Accordingly, the pre-treatment of sample and devices exerted on measuring system can be minimized and a measurement of higher accuracy can be practised.